The present invention pertains generally to a measurement apparatus including a diode laser. More particularly, the instant invention relates to a measurement apparatus including a diode laser oriented to provide an improved beam profile.
In vitro diagnostic assays have been performed with microspheres for over twenty years. The microspheres include microparticles, beads, polystyrene beads, microbeads, latex particles, latex beads, fluorescent beads, fluorescent particles, colored particles and colored beads. The microspheres serve as vehicles for molecular reactions. Microspheres for use in flow cytometry are obtained from manufacturers, such as Luminex Corp. of Austin, Tex.
Illustrative microspheres and methods of manufacturing same are, for example, found in U.S. patent application Ser. No. 09/234,841 to Mark B. Chandler and Don J. Chandler, entitled Microparticles with Multiple Fluorescent Signals, and in U.S. patent application Ser. No. 09/172,174 to Don J. Chandler, Van S. Chandler, and Beth Lambert, entitled Precision Fluorescently Dyed Particles and Methods of Making and Using Same, both patent applications incorporated herein by reference in their entirety. By way of example, if a user were performing an Ig G, A, M Isotyping Assay, the user opts for bead sets, such as Luminex 8070 IgG, 8060 IgA, and 8050 IgM bead sets.
Microspheres or beads range in diameter from 10 nanometers to 100 microns and are uniform and highly spherical. Bead-based assays are embodied in a standard xe2x80x9cstrip test,xe2x80x9d where beads coated with a capture reactant are fixed to a location on a paper strip and beads with another reactant occupy another position on the same paper strip. When a target analyte is introduced to the strip, the first bead type attaches to it and flows or mixes with the second, often causing a color change which indicates the presence of the target analyte.
More recent bead-based assays use flow cytometry to measure reactions with target analytes of interest. In conventional flow cytometers, as shown in FIG. 1, sample biological fluid containing sample cells or microspheres having reactants on their surfaces is introduced from a sample tube into the center of a stream of sheath fluid. The sample fluid stream is injected into, at, or near, the center of the flow cell or cuvette 1910. This process, known as hydrodynamic focusing, allows the cells to be delivered reproducibly to the center of the measuring point. Typically, the cells or microspheres are in suspension in the flow cell.
A laser diode 1900 focuses a laser beam on them as they pass through the laser beam by a flow of a stream of the suspension. Laser diodes in conventional flow cytometers often require shaping a round beam into an elliptical beam to be focused on the flow cell 1910. As shown in FIG. 1, this elliptical beam is often formed from the round beam using beam shaping optics 1960 located between the laser diode 1900 and the flow cell 1910.
When an object of interest in the flow stream is struck by the laser beam, certain signals are picked up by detectors. These signals include forward light scatter intensity and side light scatter intensity. In the flow cytometers, as shown in FIG. 1, light scatter detectors 1930, 1932 are located opposite the laser diode 1900, relative to the flow cell 1910, to measure forward light scatter intensity, and to one side of the laser, aligned with the fluid-flow/laser beam intersection to measure side scatter light intensity. Forward light scatter intensity provides information concerning the size of individual cells, whereas side light scatter intensity provides information regarding the relative size and refractive property of individual cells.
Known flow cytometers, such as disclosed in U.S. Pat. No. 4,284,412 to HANSEN et al., incorporated herein by reference, have been used, for example, to automatically identify subclasses of blood cells. The identification was based on antigenic determinants on the cell surface which react to antibodies which fluoresce. The sample is illuminated by a focused coherent light and forward light scatter, right angle light scatter, and fluorescence are detected and used to identify the cells.
As described in U.S. Pat. No. 5,747,349 to VAN DEN ENGH et al., incorporated herein by reference, some flow cytometers use fluorescent microspheres, which are beads impregnated with a fluorescent dye. Surfaces of the microspheres are coated with a tag that is attracted to a receptor on a cell, an antigen, an antibody, or the like in the sample fluid. So, the microspheres, having fluorescent dyes, bind specifically to cellular constituents. Often two or more dyes are used simultaneously, each dye being responsible for detecting a specific condition.
Typically, the dye is excited by the laser beam from a laser diode 1900, and then emits light at a longer wavelength. FIG. 1 depicts a prior art flow cytometer which uses beam splitters 1942, 1944, 1946 to direct light from the flow cell 1910 to photo-multiplier and filter sets 1956, 1958, 1959 and to side light scatter detector 1932. This flow cytometer employs a mirror 1970 to reflect forward light scatter to forward light scatter detector 1930.
In a standard flow cytometric competitive inhibition assay, by way of example, an antibody is covalently bound to microspheres. These beads are mixed with a biological sample along with a fluorescenated antigen. In the presence of an antigen of interest, the fluorescenated antigen competes for space on the beads, while in its absence, the fluorescenated antigen envelops the bead. Upon examination by flow cytometry, the presence of the antigen of interest is indicated by a marked decrease in fluorescence emission relative to a sample which contains the antigen of interest.
I have determined that there is, however, a stark contrast between these two types of bead-based assays. The former is simple and inexpensive, but is limited to crude assays with strong sample. concentrations of the analyte of interest. The latter is powerful and highly sensitive, but requires a $100,000 instrument and a highly trained technician to run the assay and interpret the results.
I have recognized that there is no commercially available instrument that bridges the gap between these two types of bead-based assays. I have determined that an apparatus that combines the sensitivity and flexibility of flow cytometric assays with the simplicity and low cost of strip assays would advance the art of in vitro diagnostics.
I have recognized that much of the cost and size of a flow cytometer is attributable to the laser. Virtually all commercial flow cytometers use an argon ion 488 nm laser as an excitation source. It is large, occupying several cubic feet, requires a massive power supply, and needs constant forced air cooling to maintain stability. There are other smaller and less expensive lasers, but I have ascertained that they are unsuitable for flow cytometry. For example, dye lasers burn out too quickly. He-Cd lasers are too noisy. Frequency doubled lasers are too weak. The He-Ne laser is reasonably effective, but its red output is not the color of choice in flow cytometry.
In view of the shortcomings of the above-mentioned lasers, I have assessed the merits of laser diodes. However, I have determined that the problem with diode lasers is their beam profiles. FIG. 2a, by way of example, shows a sample beam profile of a standard laser diode. The beam profile of the laser diode is very uneven as compared to that of a standard argon ion laser, as shown, by way of example, in FIG. 2b. 
I have recognized that the unevenness presents a significant obstacle for flow analyzers because associated fluorescence measurements depend upon substantially uniform excitation among particles and cells. This obstacle can be explained with reference to FIG. 3, which shows, by way of example, a two-dimensional graph of a major axis of the laser diode beam profile depicted in FIG. 2a. I have determined that if the major axis of the beam profile of FIG. 3 lies across a flow path of a flow analyzer, objects in the flow stream, such as cells or microspheres are not subject to light having the same or substantially the same energy levels. Rather, as shown in FIG. 3, points 10, 12, 14, 16, and 18 on the graph have energy levels that vary indiscriminately across the beam profile.
I have determined that if a microsphere is passing through the flow stream and subject to the laser diode beam at, for example, point 10 on the graph of the beam profile would get much more energy than, if the same microsphere were passing through the flow stream and subject to the laser diode beam at point 14. As such, I have recognized that it is impossible to distinguish between a microsphere having a high fluorescence intensity passing through a point on the beam profile having a low energy level or a microsphere having a low fluorescence intensity passing through a point on the beam profile having a high fluorescence intensity.
Commercial flow cytometers, that offer diode lasers as a second laser to accompany the argon ion laser, take for granted the large coefficients of variation (CVs) of the beam profile of the diode laser. Moreover, laser diodes need not have identical beam profiles. Indeed, even minor differences in resonating cavities, for example, affect the shape of respective beam profiles. Thus, a diode laser in a flow cytometer of a given model need not have the same beam profile of a diode laser in another flow cytometer of the same model.
As such, commercial flow cytometers, as shown by way of example, in FIG. 1, employ beam shaping optics, such as prismatic expanders, beam shaping expanders, and micro lens arrays. Prior art implementations of diode lasers in flow cytometry have attempted to optically correct the beam, steering the two outside peaks toward the center.
I have determined that such optics are unnecessarily expensive by themselves, and add to the manufacturing complexity of the flow cytometers, which, in turn, further adds to the overall cost of the instrument. Moreover, I have determined that despite the expensive and complex beam shaping optics employed, the resulting beam profile is still unsatisfactory, as shown in FIG. 4. Although the beam profile in FIG. 4 is better than that shown in FIG. 2a, for example, it still yields a ten to fifteen percent variation in energy intensity across the flow path.
In view of the above, I have determined that it would be desirable to have a method and/or apparatus for providing precise measurements of light scatter and fluorescence by accommodating an uneven beam profile of a diode laser.
I have also determined that it would be desirable to have such a method and/or system absent beam shaping optics optically cooperating with or coupled to the laser diode.
I have further determined that it would be desirable to have such a method and/or system including a flow analyzer.
It is a feature and advantage of the instant invention to provide a method and/or apparatus for providing precise measurements of light scatter and fluorescence by accommodating an uneven beam profile of a light source, such as a laser diode.
It is also a feature and advantage of the instant invention to provide such a method and/or system absent beam shaping optics optically cooperating with or coupled to the laser diode.
It is also a feature and advantage of the instant invention to provide such a method and/or system including a flow analyzer to achieve precise measurements of light scatter and fluorescence emitted by microspheres or beads.
It is a feature and advantage of the instant invention to provide a novel diagnostic system. The instant diagnostic system includes a measurement device including a flow path and a light source, such as a laser diode, and communicatable with a computer. The light source includes a Gaussian first beam profile across the flow path and a second beam profile along the flow path. The diagnostic system further includes a memory medium readable by the computer and storing computer instructions executable by the computer. The instructions include the following sequential, non-sequential, or independent steps. A template relating to a beam profile of the light source is built. A fluorescent sample is captured by the measurement device. The sample is time-wise aligned to the template. The sample is normalized relative to the template. The normalized sample is integrated to determine a total amount of fluorescence in the sample.
Optionally, the template relates to a microsphere size and/or a flow rate. Optionally, the time-wise aligning step includes applying a least squares method for alignment.
Optionally, the measurement device includes a flow analyzer. The flow analyzer is optionally free of a beam profile shaping element optically cooperating with the light source, such as a prismatic expander, a micro lens array, and a beam expander. Optionally, the flow analyzer, in operation, includes a flow path, the beam profile of the light source having a major axis aligned with the flow path. Optionally, the flow analyzer is free of a peak detector for detecting a fluorescence intensity peak for the sample event.
It is also a feature and advantage of the instant invention to provide a computer program product for use with a computer and a measurement device including a light source having a first beam profile and a fluid flow path subject thereto. The computer program product includes a memory medium readable by the computer and storing computer instructions. The instructions include the following sequential, non-sequential, or independent steps. A template relating to the first beam profile of the light source along the flow path is built. The light source includes a Gaussian second beam profile across the flow path. A fluorescent sample is captured by the measurement device. The sample is time-wise aligned to the template. The sample is normalized relative to the template. The normalized sample is integrated to determine a total amount of fluorescence in the sample.
Optionally, the template relates to a microsphere size and/or a flow rate. Optionally, the time-wise aligning step includes applying a least squares method for alignment.
It is another feature and advantage of the instant invention to provide a method of improving a beam profile of a light source, such as a laser diode, in a measurement device. The measurement device includes a flow path and a light source having a Gaussian beam profile across the flow path and a second beam profile along the flow path. The instant method includes the following sequential, non-sequential, or independent steps. A template relating to a beam profile of the light source is built. A fluorescent sample is captured by the measurement device. The sample is time-wise aligned to the template. The sample is normalized relative to the template. The normalized sample is integrated to determine a total amount of fluorescence in the sample.
Optionally, the template relates to a microsphere size and/or a flow rate. Optionally, the time-wise aligning step includes applying a least squares method for alignment.
It is yet another feature and advantage of the instant invention to provide a flow analyzer including a flow cell defining a flow path. The flow analyzer further includes one or more light sources, such as laser diodes, including a Gaussian first beam profile across the flow path and a second beam profile along the flow path. Optionally, the flow analyzer is free of a beam shaping optical element or assembly optically cooperating with one or more of the light sources.
Optionally, the flow analyzer is free of a peak detector for detecting a fluorescence intensity peak of a sample event. Optionally, the flow analyzer further includes one or more optical detectors cooperating with the one or more laser diodes and the flow cell. The one or more optical detectors include an avalanche photodiode, a photomultiplier tube, or a p-i-n photodiode.
Optionally, the flow analyzer further includes one or more analog-to-digital converters communicating with a respective optical detector. The flow analyzer optionally also includes one or more digital signal processor controlling the one or more analog-to-digital converters.
It is another feature and advantage of the instant invention to include, in a flow analyzer including one or more light sources and a flow cell defining a flow path, a method of improving a beam profile characteristic. The method includes orienting the one or more light sources relative to the flow cell so that the one or more light sources includes a Gaussian first beam profile across the flow path and a non-Gaussian second beam profile along the flow path.
Optionally, in the novel method, the flow analyzer is free of a beam shaping element or assembly optically coupled to the one or more light sources. Optionally, the one or more light sources includes one or more laser diodes.
There has thus been outlined, rather broadly, the more important features of the invention in order that the detailed description thereof that follows may be better understood, and in order that the present contribution to the art may be better appreciated. There are, of course, additional features of the invention that will be described hereinafter and which will form the subject matter of the claims appended hereto.
In this respect, before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting.
As such, those skilled in the art will appreciate that the conception, upon which this disclosure is based, may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.
Further, the purpose of the foregoing abstract is to enable the U.S. Patent and Trademark Office and the public generally, and especially the scientists, engineers and practitioners in the art who are not familiar with patent or legal terms or phraseology, to determine quickly from a cursory inspection the nature and essence of the technical disclosure of the application. The abstract is neither intended to define the invention of the application, which is measured by the claims, nor is it intended to be limiting as to the scope of the invention in any way.
These together with other objects of the invention, along with the various features of novelty which characterize the invention, are pointed out with particularity in the claims annexed to and forming a part of this disclosure. For a better understanding of the invention, its operating advantages and the specific objects attained by its uses, reference should be had to the accompanying drawings and descriptive matter in which there is illustrated preferred embodiments of the invention.
The detailed descriptions which follow may be presented in terms of program procedures executed on a computer or network of computers. These procedural descriptions and representations are the means used by those skilled in the art to most effectively convey the substance of their work to others skilled in the art.
A procedure is here, and generally, conceived to be a self-consistent sequence of steps leading to a desired result. These steps are those requiring physical manipulations of physical quantities. Usually, though not necessarily, these quantities take the form of electrical or magnetic signals capable of being stored, transferred, combined, compared and otherwise manipulated. It proves convenient at times, principally for reasons of common usage, to refer to these signals as bits, values, elements, symbols, characters, terms, numbers, or the like. It should be noted, however, that all of these and similar terms are to be associated with the appropriate physical quantities and are merely convenient labels applied to these quantities.
Further, the manipulations performed are often referred to in terms, such as adding or comparing, which are commonly associated with mental operations performed by a human operator. No such capability of a human operator is necessary, or desirable in most cases, in any of the operations described herein which form part of the present invention; the operations are machine operations. Useful machines for performing the operation of the present invention include general purpose digital computers or similar devices.